The freeze-etch electron microscopic techniques developed in this laboratory will be used to study clathrin-mediated endocytosis in fibroblasts and macrophages. Questions to be addressed include: (1) what role do clathrin lattices play in bringing about receptor clustering? (2) what is the mechanism by which clathrin lattices curve into spheres and pinch off from the plasma membrane? and (3) how is clathrin recycled from internalized membranes back to the cell surface for additional round of endocytosis? Ongoing studies of all three processes are described to demonstrate the unique contributions that the quick-freeze, deep-etch, rotary=replication technique can make to this analysis. By permitting direct visualization of individual membrane receptors on the outsides of cells, as well as individual clathrin lattices on their insides, the technique has already demonstrated a number of correlations between clathrin lattice dynamics and changes in receptor distribution. By also permitting 3-D visualization of small clathrin polymers and individual clathrin """"""""triskelia"""""""" adsorbed to mica, the technique has also begun to shed light on the molecular interactions that underlie clathrin lattice dynamics. A broad range of experiments is proposed to further probe the rules by which clathrin molecules self-assemble and carry out their biological functions. Some of these experiments will involve electron microscopic analysis of the effects of chemical treatments that stimulate or inhibit endocytosis in cultured cells, particularly the inhibitory effects of cytoplasmic acidification and the stimulatory effects of cytoplasmic alkalinization. In addition, new observations on the effects of such pH changes on endosome and lysosome movement and fusion will be actively pursued. Finally, the molecular basis of clathrin lattice dynamics will be analyzed by observing the patterns of molecular interaction that clathrin displays with itself and with other proteins in vitro, under different conditions that mimic the above in vivo situations. These studies will hopefully increase our basic understanding of how cells regulate their uptake of proteins and their intracellular membrane traffic.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM029647-16
Application #
2838472
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1981-12-01
Project End
2000-11-30
Budget Start
1998-12-01
Budget End
1999-11-30
Support Year
16
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Washington University
Department
Physiology
Type
Schools of Medicine
DUNS #
062761671
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Heuser, John E (2011) The origins and evolution of freeze-etch electron microscopy. J Electron Microsc (Tokyo) 60 Suppl 1:S3-29
Huang, Jing; Roth, Robyn; Heuser, John E et al. (2009) Integrin alpha(v)beta(3) on human endothelial cells binds von Willebrand factor strings under fluid shear stress. Blood 113:1589-97
Hanson, Phyllis I; Roth, Robyn; Lin, Yuan et al. (2008) Plasma membrane deformation by circular arrays of ESCRT-III protein filaments. J Cell Biol 180:389-402
Huang, Ren-Huai; Wang, Ying; Roth, Robyn et al. (2008) Assembly of Weibel-Palade body-like tubules from N-terminal domains of von Willebrand factor. Proc Natl Acad Sci U S A 105:482-7
Cardone, Giovanni; Winkler, Dennis C; Trus, Benes L et al. (2007) Visualization of the herpes simplex virus portal in situ by cryo-electron tomography. Virology 361:426-34
McCarren, J; Heuser, J; Roth, R et al. (2005) Inactivation of swmA results in the loss of an outer cell layer in a swimming synechococcus strain. J Bacteriol 187:224-30
Heuser, John (2005) Deep-etch EM reveals that the early poxvirus envelope is a single membrane bilayer stabilized by a geodetic ""honeycomb"" surface coat. J Cell Biol 169:269-83
Szajner, Patricia; Weisberg, Andrea S; Lebowitz, Jacob et al. (2005) External scaffold of spherical immature poxvirus particles is made of protein trimers, forming a honeycomb lattice. J Cell Biol 170:971-81
Bretschneider, Till; Diez, Stefan; Anderson, Kurt et al. (2004) Dynamic actin patterns and Arp2/3 assembly at the substrate-attached surface of motile cells. Curr Biol 14:1-10
Engqvist-Goldstein, Asa E Y; Zhang, Claire X; Carreno, Sebastien et al. (2004) RNAi-mediated Hip1R silencing results in stable association between the endocytic machinery and the actin assembly machinery. Mol Biol Cell 15:1666-79

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